Regulation of the enzootic cycle in Borrelia burgdorferi requires a shift to the RNA polymerase alternative sigma factor, RpoS. We used in vitro and in vivo assays to assess the relative importance of the putative Shine-Dalgarno sequence and its sequestration for the translational efficiency of rpoS. We created mutant leader regions in which we either removed the Shine-Dalgarno sequence, disrupted the secondary structure or both. Binding assays and toeprint assays demonstrated that both the presence and the availability of the Shine-Dalgarno sequence are important to the efficiency and specificity of ribosome binding. Adding a DsrA(Bb) mimic in the form of a single-stranded DNA oligonucleotide increased the level and specificity of binding ribosomes to the transcript with an extended leader, presumably by making the Shine-Dalgarno sequence available for binding. In in vivo assays we confirmed that the Shine-Dalgarno sequence must be both present and un-sequestered in order for translation to proceed efficiently. The longer transcript was significantly better translated in B. burgdorferi at 37 degrees C than at 26 degrees C, lending support to the hypothesis that DsrA(Bb) acts as a temperature-dependent stimulator of translation. These studies demonstrate that translational regulation of gene expression in B. burgdorferi may be an important mechanism for responding to environmental signals important in the enzootic cycle. (c) 2013 Elsevier Inc. All rights reserved.